Hepatitis C virus (HCV) is a leading cause of cirrhosis and hepatocellular carcinoma worldwide, with an estimated 185 million people infected and over 350,000 deaths a year. In developed countries, injection drug use is the predominant route of transmission and accounts for over two thirds of the HCV infections in the U.S., with up to 80% of injection drug users infected with HCV. Although new direct acting antiviral drugs will play an important role in controlling HCV, treatment is not likely to reduce global prevalence due to the high cost and because most people are subclinically infected and will not seek treatment. A protective vaccine would be much more cost-effective, but the correlates of protective immunity are not completely understood. Importantly, HCV only infects humans and chimpanzees, and because research on chimpanzees has been phased out, mechanisms of protective immunity must be rigorously dissected in an alternative animal model. Recently, a novel nonprimate hepacivirus has been described in horses. This equine hepacivirus (EHCV) is hepatotropic and is the closest known phylogenetic relative to HCV. The overall goal of the proposed studies is to delineate the protective roles of viral-specific memory T cells and antibodies against hepaciviral infection. The hypothesis to be tested is that horses that resolve EHCV infection mount broadly active memory T cell and/or antibody responses that are sufficient to control homologous and heterologous EHCV replication. We will use our established methods to first identify EHCV proteins containing conserved epitopes recognized by memory T cells from horses that have spontaneously resolved primary EHCV infection. Memory CD4+ T cell lines with high EHCV-specific proliferative responses will be expanded in culture, as will EHCV-specific IFN? producing memory CD8+ T cell lines with cytotoxic activity. These T cells will then be transferred into MHC-matched horses with severe combined immunodeficiency (SCID) and protective effects against a mixed EHCV challenge determined. We will then similarly determine if antibodies that broadly inhibit EHCV E2 glycoprotein binding to equine CD81 are protective against EHCV challenge. These proposed studies will definitively demonstrate whether or not virus-specific T cells and/or antibodies can independently control EHCV infection and will have critical implications for HCV vaccine design, where experiments of this type are not possible.